ABSTRACT The goals of this study are to determine the function and mechanism of two non-coding microRNAs in regulating osteogenesis in vitro and also in vivo in the context of bone repair/disease. MicroRNAs (miRNAs) are small, non- coding epigenetic regulators that target and suppress translation of numerous mRNAs within a given cell type, resulting in modulation of many pathways and networks. We previously reported on differentially-expressed miRNAs within the human embryonic growth plate of developing long bones and identified two miRNAs (miR- 181a-1 and miR-138) that were more highly expressed in hypertrophic chondrocytes compared to progenitor chondrocytes, suggesting functional roles in regulating chondrogenesis and/or endochondral ossification. We subsequently found that these miRNAs have opposing roles in regulating osteogenesis in vitro: miR-181a/b-1 (miR-181a-1 and its clustered miRNA, miR-181b-1) enhances while miR-138 inhibits this process. We also found that miR-181a/b-1 enhances PI3K/AKT signaling and mitochondrial metabolism. New preliminary data suggests that the mitochondrial enzyme, pyruvate dehydrogenase 4 (PDK4) is a potential target gene of miR-181a/b-1. PDK4 functions to inhibit the pyruvate dehydrogenase complex (PDC) resulting in decreased mitochondrial metabolism. Thus, suppression of PDK4 by miR-181a/b-1 may partly explain the enhancing effects of this miRNA cluster on mitochondrial respiration and osteogenesis. In Specific Aim 1, we therefore plan to test if miR-181a/b- 1 suppresses PDK4 directly and/or indirectly via the PI3K/AKT/FoxO1 pathway (given that FoxO1 is a known transcriptional activator of PDK4). We also plan to test if miR-138 has opposing effects on PDK4 expression since we found that this miRNA suppresses PI3K/AKT signaling as well as pathways associated with oxidative phosphorylation. This aim will also explore the effects of two PDK4 inhibitor drugs on potentially enhancing osteogenesis. Utilizing murine models of heterotopic ossification (HO) and bone fracture, we found that miR-138 over-expression suppresses HO formation and non-endochondral bone fracture repair while miR-181a/b-1 over- expression enhances endochondral bone fracture healing. These findings highlight that the in vitro function of both miRNAs is translatable in vivo in repair/disease models involving new bone formation. Therefore, Specific Aims 2 and 3 will investigate the effects of modulating miR-181a/b-1 or miR-138 in vivo to attempt to suppress HO and enhance fracture healing, respectively. Effects of PDK4 inhibitor drugs will also be tested in the fracture models depending on findings from Aim 1. Overall, these studies are designed to test our overall hypothesis that appropriate targeting of miR-181a/b-1 or miR-138 in vivo will modulate bone formation during HO or bone repair and that these effects are due, in part, to regulation of PI3K/AKT/FoxO1/PDK4 signaling and mitochondrial metabolism. These studies are important because new mechanistic information will be gained on how these miRNAs regulate mitochondrial respiration during osteogenesis. In addition the potential therapeutic value of targeting each miRNA as a means to treat bone fractures or HO will be determined.